This invention relates generally to optical microscopy and in particular to reflected-light (epi-illumination) wide-field microscopy.
In a conventional non-scanning (wide-field) optical microscope, radiation (typically visible light, ultraviolet light or x-rays) interacts (e.g. by reflection, refraction, or diffraction) with a specimen located at the object plane of the microscope optics, to create a pattern that is processed by the microscope to create an image that is enlarged to a size that is many times greater than the specimen itself. In a conventional reflected-light wide-field compound refractive microscope, radiation reflected from the specimen passes through an objective that focuses the radiation into an enlarged real image on an intermediate image plane. The intermediate image is then additionally magnified by an eye lens which produces an enlarged real or virtual image depending on the particular application.
It is well-know that at very high magnifications optical microscopes will exhibit the characteristic that point objects are seen as fuzzy disks surrounded by diffraction rings. The resolving power of a microscope is taken as the ability of the microscope to reveal adjacent structural detail as distinct and separate. Even assuming perfect refraction by the lens system of the microscope, the minimum size of the diffraction rings, which determines the resolution (d) of the microscope is still limited function by both the wavelength of light (λ), and the numerical aperture (NA) of the objective lens as expressed by the following equation.
  d  =      λ          2      ⁢      NA      
In practice, the highest numerical aperture that can be achieved in air is about 0.95. Therefore and with green light (about 550 nm wavelength) the diffraction limit of a conventional optical microscope is about 200 nm (the Abbe Limit).
Numerous methods have been successfully employed over the years in order to gain ever increasing magnification and resolution, including use of ultraviolet light and x-rays (shorter wavelength) and oil immersion (increased numerical aperture). These methods, however, are still limited by the Abbe limit of the optical system. A technique for increasing resolution beyond the Abbe limit of an optical system is laser scanning confocal microscopy. In a laser confocal microscope, a laser beam passes through a light source pinhole aperture and beam splitter and is focused by an objective lens onto the specimen. The light reflected from the specimen is focused through the objective lens onto the beam splitter, which reflects the light onto a photo-detector through a pinhole aperture. The pinhole aperture blocks any reflected light that is not emanating from the focal point of the objective (which would be out of focus). Laser confocal microscopes operate above the Abbe limit of the optical system but do not produce a wide-field image. Consequently the image must be constructed by scanning the sample point by point.